Molecular cloning of Zangptl1, Zangptl2 and Zangptl6. (A) Percentages of amino acid identity of Zangptl1, Zangptl2 and Zangptl6 to human (upper panel), and mouse (lower panel) counterparts. (B) RT-PCR analysis of Zangptl1, Zangptl2 and Zangptl6 expression in adult tissues. Zangptl1 was expressed broadly in various tissues and abundantly in spleen, muscle and gut. Zangptl2 was abundantly expressed in heart and skeletal muscle. Zangptl6 was predominantly expressed in liver and gut. β-Actin expression served as an internal control of template cDNAs. (C) Percentages of amino acid similarities of Zangptl6 to human Ang1, 2 and 4, mouse Ang3 and human Angptl3, 4 and 5. No other mammalian Angs or other Angptls were more closely related to Zangptl6 than mammalian Angptl1, Angptl2 and Angptl6. (D) Phylogenetic relationships among Angptls and Angs. The tree was constructed by using the Megalign program of DNAstar (based on Jotun Hein Method). h, human; m, mouse. Bar indicates 1000 nucleotides.

Embryonic expression of Zangptl1. Whole-mount in situ hybridization was done with a Zangptl (A–D) probe or a myoD probe (E–F) at 24hpf. Weak expression in the somites of a 24hpf embryo especially at the middle portion of tail (arrows in A) was detected. Higher magnification dorsal–ventral (B) and lateral views (C) of a 24hpf embryo are shown. Arrowheads indicate Zangptl1 expression in the somites. (D) A sagittal section of a 24hpf embryo at the middle portion of tail. As comparison of Zangptl1 expression, dorsal–ventral (E) and lateral views (F) of a 24hpf embryo stained with myoD are shown. Arrowheads, somites; sc, spinal cord; nc, notochord. Bar indicates 50 μm.

Embryonic expression of Zangptl2. Whole-mount in situ hybridization was done with a Zangptl2 probe at 18hpf (A,D), 24hpf (B,E–H) and 48hpf (C,I,J), and with a foxA1 probe at 48hpf (K). In an 18hpf embryo (A), Zangptl2 expression was detected in the yolk sac extension and spinal cord. A sagittal sections of a 18hpf embryo are shown in (D). In a 24hpf embryo (B), the expression intensity became stronger and was evident not only in the yolk sac extension and spinal cord, but in branchial arches. Higher magnification lateral (E) and dorsal–ventral views (H) of a 24hpf embryo are shown. Dotted and solid lines in (E) denote the level of sagittal sections shown in (F) and (G), respectively. In a 48hpf embryo, Zangptl2 expression was detected in the liver primordium and in pectoral fin buds (C,I,J). A 48hpf embryo stained with foxA1 known as a marker of liver primordium and gut is shown in (K). Closed arrowheads, spinal cord; closed arrows, yolk sac extension; open arrowheads, branchial arches; open arrows, pectoral fin bud; asterisk, liver primordium; double asterisk, gut. Bars indicate 50 μm.

Embryonic expression of Zangptl6. Whole-mount in situ hybridization was done with a Zangptl6 probe at 14hpf (A), 18hpf (B) and 24hpf (C–E) and with a shh probe at 24hpf (F,G). In an 14hpf embryo (A), Zangptl6 expression was seen in almost entire length of notochord. In a 18hpf (B) and 24hpf (C) embryos, Zangptl6 expression was restricted to the caudal end of notochord. Higher magnification of a 24hpf embryo is shown in (D). In sagittal sections of a 24hpf embryo (E), Zangptl6 expression was detected in the notochord ventral to the spinal cord. High magnification lateral view (F) and a sagittal section (G) of a 24hpf embryo stained with shh as a marker of notochord are shown. Closed arrowheads, notochord; sc, spinal cord. Bars indicate 50 μm.

Zangptl2 expression in adult fin regeneration. Whole-mount in situ hybridization with a Zangptl2 probe was performed on regenerated fins at 0hpa (A), 12hpa (B), 24hpa (C), 48hpa (D) and 96hpa (E). (F) A longitudinal section of a stained fin shows Zangptl2 expression in the blastema under the regenerated epidermis. (G) A longitudinal section of a fin stained with msxb as a marker of blastema is shown. (H) RT-PCR analysis of regenerated fins revealed induction of Zangptl2 expression during the course of fin regeneration. Fins stained with shh as known to be expressed in basal layer of the epidermis in proximity to blastema at 48hpf (I) and 96hpf (J) are shown. Experiments were also performed in water containing DMSO (K) or 10 μM of the Fgfr inhibitor SU5402 (fgfRi) (L,M), and stained with Zangptl2. Zangptl2 expression was unchanged following treatment with DMSO alone at 24hpa (K), but was not detected when fish were treated with SU5402 at 24hpa (L) and 48hpa (M). Closed arrowheads, blastema; open arrowheads, basal layer of the epidermis in close proximity to blastema. Bars indicate 50 μm.